ABSTRACT A major force driving relapse in drug addiction, including opiate addiction, is the almost intolerable aversive effects of drug withdrawal. The biological bases of learned aversion to the drug withdrawal state have important mechanistic overlap with those for aversion to non-drug-related noxious stimuli (e.g. bitter taste, footshock, social defeat, traumatic stress) and involve multiple brain regions. Among them, the interconnected basal ganglia (BG) nuclei, including nucleus accumbens (NAc), ventral pallidum (VP) and dopaminergic (DA) neurons in the ventral tegmental areas (VTA) appear to be particularly crucial. In this proposal, we will focus our study on the NAc, not only because prior studies have implicated this brain region as a critical substrate for both rewarding and aversive effects of opiates, but also because recent studies suggest a particular neuronal cell type in the NAc, the D2 medium spiny neuron (D2-MSN), is essential to establishment of avoidance behaviors elicited by noxious environmental stimuli as well as the intense aversive effects of opiate withdrawal. Since manipulating the activities of D2-MSNs using the latest chemogenetic or optogenetic tools have demonstrated pivotal roles of these neurons in aversive behaviors, we reason the critical next step is to dissect the roles of D2-MSNs in non-drug-related and opiate withdrawal-related aversion through genetic analyses of endogenous genes that are highly selectively expressed in D2-MSNs. To this end, we hypothesize three such genes - two receptors (Gpr6 and Adora2a) and one peptide precursor (pre-pro-enkephalin (Penk)) - play important roles in opiate withdrawal aversion. We propose that signaling through these two Gs-linked receptors, by opposing the signaling imparted by the Gi/o-coupled dopamine D2 receptor, is crucial for D2- MSN mediation of both non-drug and opiate withdrawal-related aversion. To test this, we will investigate the behavioral and signaling effects of single or double deletion of these receptors. Conversely, our recent data demonstrate that pro-enkephalin-derived peptides in D2-MSNs play a crucial role in mediating an endogenous opioid hedonic tone, hence we will test the idea that removal of Penk in D2-MSNs will exacerbate the aversive effects of opiate withdrawal. Finally, we have obtained exciting preliminary data, using RNA-seq and network analyses, showing aberrant changes in NAc molecular networks upon the establishment of opiate dependence. We will apply such powerful integrative systems biology approaches to investigate whether genetic perturbation of key D2-MSN genes or opioid network genes alters the molecular signatures of opiate dependence.